Merkel cell polyomavirus T antigens and neoplastic transformation in vivo Project Summary Merkel cell carcinoma (MCC) is a rare neuroendocrine skin tumor with a poor prognosis at advanced disease stages due to the unavailability of effective treatments. Most MCCs carry sequences from a novel polyomavirus, Merkel cell polyomavirus (MCPyV), and express two putative oncoproteins: MCPyV small T antigen (sTAg) and tumor-specific truncated large T antigen (tLTAg). Like other viral oncoproteins, MCPyV transforming antigens are predicted to interact with multiple cellular proteins including tumor suppressors: tLTAg targets RB1 while sTAg targets the PP2A complex. At least some knock-down studies in cultured cells and xenografts support a requirement for tLTAg and sTAg antigens in MCC tumor cell maintenance. In addition, overexpression studies in cultured fibroblasts point to stAg as the predominant transforming oncogene which, surprisingly, operates in a PP2A-independent manner. In contrast, sTAg-driven fibroblast transformation is strictly dependent on a recently-described domain that binds the Fbxw7 component of the SCF E3 ubiquitin ligase complex and leads to accumulation of LTAg and several cellular oncoproteins that are Fbxw7 substrates. These reports have provided valuable insight into the transforming potential of sTAg in cultured fibroblasts, but studies assessing whether MCPyV TAgs can function as oncogenic drivers in vivo have not yet been reported. In preliminary studies we show that MCPyV sTAg, but neither tLTAg nor full-length LTAg, is a potent transforming oncogene in skin and oral epithelia of transgenic mice, leading to striking hyperplasia, impaired terminal differentiation, a DNA damage response, and apoptosis. We propose a series of experiments to further study the in vivo transformation potential of individual MCPyV TAgs, and to test their functional interaction when co-expressed in inducible mouse models. To investigate the MCC tumor cell of origin, we will compare MCPyV TAg transforming potential when targeted to 1) proliferating versus differentiating cellular compartments of epidermis, and 2) Merkel cell progenitors versus differentiated Merkel cells. We will also isolate defined cell populations from conditional MCPyV TAg transgenic mice and study their response to TAg expression in cell culture, enabling functional studies aimed at defining mechanisms of transformation which can then be verified in vivo. The proposed studies are highly significant since they will help fill a critical gap in our knowledge by defining the biological activity of MCPyV TAgs in intact animals, providing a much-needed set of tools for studying factors contributing to the development and maintenance of MCPyV-associated cancer. In addition, these studies will help identify MCPyV TAg cellular targets whose deregulation via non-viral mechanisms may drive the development of virus-negative MCC, and may contribute more generally to the development of other types of cancer. Finally, the proposed work is likely to have direct translational relevance to MCC patients, as it may lead to the identification of new therapeutic targets and yield much-needed mouse models for functional studies and preclinical trials.